The overall objective of this proposal is to elucidate certain aspects of the basic biochemical and metabolic events involved with the initiation and regulation of macrophage activation by the immunopotentiator maleic vinyl ether (MVE). Murine peritoneal cells enriched for macrophages by adherence to fetal calf serum substration and fractionated into macrophage subpopulations by centrifugal elutriation will be used as the source of macrophages. The state of macrophage activation by in vitro exposure to MVE will be assessed by employing two macrophage-mediated tumor cytotoxicity assays, an 125IUdR incorporation assay to measure tumor cytostasis, and a soft agar cloning assay to measure tumor cytolysis. To ascertain the underlying biochemical mechanism of macrophage activation, measurement of the intracellular modulators cyclic AMP (cAMP), cyclic GMP (cGMP), calcium and magnesium will be performed before, during, and after exposure to MVE. The cyclic nucleotide levels will be monitored by radioimmunoassay whereas the metabolism of divalent cations during macrophage activation will be determined using the fluorescent chelate probe chlorotetracycline. In the event that alterations in cyclic nucleotides and/or divalent cation metabolism temporally correlate with the activation process, pharmacologic probes which alter endogenous levels of cyclic nucleotides or perturb cellular calcium metabolism will be employed to ascertain whether these changes are essential to or coincident with macrophage activation. If the results of these experiments suggest that Ca ion 2 mobilization is necessary for macrophage activation, additional studies using 45Ca ion 2 labeling techniques will be conducted in an attempt to identify the intracellular calcium compartment(s) involved in the activation process and the describe quantitatively the calcium fluxes within these compartments. In the event that alterations in cyclic nucleotide levels appear to be essential to the activation process, the measurement of cyclases, phosphodiesterases and protein kinases will be performed in an effort to elucidate the mechanisms of cyclic nucleotide modulation. The results of these experiments will be correlated with changes in major biochemical events such as nucleic acid and protein biosynthesis as well as glycolysis, so that a scheme of macrophage activation by MVE may be established.